1. Field of the Invention
The present invention relates generally to the field of protein production and purification. More specifically, the present invention relates to a centrifuge apparatus for use in methods for isolating concentrated, active proteins from the intercellular material of plants via a vacuum and centrifugation process which does not destroy the plant material, permitting secondary protein extraction from the plant material.
2. Description of the Prior Art
There are many examples of valuable proteins that are useful in pharmaceutical and industrial applications. Often these molecules are required in large quantities and in partially or highly purified formulations to maintain product quality and performance. Plants are an inexpensive source of proteins, including recombinant proteins. Many have proposed the desirability of producing proteins in large amounts in plants. However, the problems associated with extracting and processing products from homogenized plant tissues as well as purifying and recovering the recombinant protein product have been recognized as substantial. Austin et al. Annals New York Academy of Science, 721:234-244 (1994). These problems represent major impediments to successful recombinant protein production in plants on a large and commercially valuable scale.
Plant cells are thought to synthesize proteins on the membranes of the endoplasmic reticulum and transport the proteins synthesized to the cell surface in secretory vesicles formed at the Golgi apparatus. A discussion of the topic is provided by Jones et al., New Phytology, 111:567-597 (1989). Significant research has been devoted to elucidating the specific mechanisms related to protein secretion for several particular proteins in specific plant tissues or cell cultures. Examples of such efforts are presented by Herbers et al., Biotechnology 13:63-66 (1995), Denecke et al., The Plant Cell 2:51-59 (1990), Melchers et al., Plant Molecular Biology 21:583-593 (1993) and Sato et al., Biochemical and Biophysical Research Communications 211 (3):909-913 (1995). In the case of proteins not secreted into the plant cell apoplasm or intercellular space, a mechanism for lysing the plant cell wall must be utilized in order to release and capture the protein of interest. Plant cells must be exposed to very high shear forces in order to break the cell walls and lyse cellular membranes to release intracellular contents. Proteins of interest, whether recombinantly produced or naturally produced by the subject plant, are thereby exposed to a hostile chemical environment and are particularly subject to oxidative and proteolytic damage due to the exposure of the product to enzymes and small molecules that were compartmentalized before homogenization of the tissue. In addition, most of the-other total cellular protein is mixed with the protein of interest creating formidable purification problems if such a cell lysis procedure is performed.
In order to use the biosynthetic capacity of plants for reliable protein production, a process to obtain specific proteins that can be secreted into the intercellular space (apoplasm) of plant tissues is desirable. Such a procedure would forego the need for homogenization. If such a procedure is performed, the fraction of plant material containing one or more proteins of interest might be obtained without homogenization. Therefore, such a procedure provides that the plant extract is enriched for the particular protein of interest, and the protein is protected from some chemical and enzymatic degradation.
Since the valuable proteins and products of interest are partitioned or secreted into the interstitial spaces, vacuum pressure facilitates the introduction of infiltration medium into the interstitial space. Similarly, various forces can be applied to remove the retained fluid. Centrifugal force of 1,000 G is effective. Using gravity, the retained fluid can be collected in a trap under vacuum. With or without vacuum infiltration of a buffer, the enzyme can be recovered by freezing the tissue, thawing and applying a physical press to recover the fluid. However, such a procedure results in an undesirable increased cellular lysis.
Genetically modified plants are a reliable source for the production of recombinant proteins. Because the biological product is accumulated under nonsterile growth conditions and the production may be scaled to the quantities desired in a relatively inexpensive manner, it is feasible to exploit a dilute but enriched source such as the interstitial fluid fraction as a source for harvesting proteins of interest on an industrial scale. A variety of proteins of interest may be harvested from recombinant plant sources, however, highly active, pharmaceutical quality enzymes, cytokines and antibodies are particularly valuable products that can be developed by this process.
U.S. Pat. No. 6,284,875 to Turpen et al., and assigned to the assignee of the present invention, discloses methods for recovering proteins from the interstitial fluid of plant tissues, which method generally includes the steps of:
(1) submerging plant tissue in a predetermined fluid having a predetermined pH;
(2) subjecting the submerged plant tissue to vacuum pressure to release the interstitial fluid with minimal damage to cell walls within the tissue; and
(3) centrifuging the plant tissue to draw interstitial fluid out of the plant tissue.
There is, however, a continuing need for improved apparatus to conduct processes like those of the Turpen et al. U.S. Pat. No. 6,284,875, and particularly there is a need for such apparatus which can be utilized on a repeated and automated basis. Many different processes involve sequential batch processing of different materials, such that cleaning of the production equipment between batches is necessary. For example, the production of patient-specific medicines requires a high speed automated process and requires the complete separation of the product of each batch processed, thus requiring a complete cleaning of the apparatus between batches.
The present invention provides a centrifuge apparatus for recovery of a material of interest from the interstitial fluid of plant tissues, which apparatus includes a rotatable centrifuge bowl for receiving plant tissue and a buffer solution. The apparatus further includes a vacuum sealable housing containing the centrifuge bowl, so that the plant tissue and buffer solution in the centrifuge bowl can be subjected to a substantially vacuum environment. The centrifuge bowl has a radially outwardly inclined outer wall so that interstitial fluid centrifuged from the plant tissue climbs the outer wall during centrifugation.
In another aspect of the invention the centrifuge apparatus includes a rotatable centrifuge bowl having a radially outer wall inclined outward so that during centrifugation, liquid in the bowl climbs the outer wall. The apparatus includes a vacuum sealed housing containing the rotatable centrifuge bowl so that a material of interest contained in the bowl and including the liquid can be subjected to vacuum.
In still another aspect of the invention the centrifuge apparatus includes a rotatable centrifuge bowl having a radially outwardly inclined outer wall. The apparatus further includes a housing containing the centrifuge bowl and at least one cleaning nozzle disposed in the housing and directed toward the centrifuge bowl for cleaning the centrifuge bowl. The at least one cleaning nozzle may include selective combinations of a first nozzle directed toward an interior of the bowl, a second nozzle directed toward a central exterior surface of the bowl, and a third nozzle directed to an annular space between the bowl and the housing.
In another aspect of the invention methods are provided for recovering a material of interest from the interstitial fluid of plant tissues, wherein the plant tissue is placed in a centrifuge, the centrifuge is sealed, a buffering solution is introduced, a vacuum is created in the centrifuge thereby allowing the buffering solution to infiltrate the plant tissue, and the centrifuge is rotated thereby drawing the buffering solution and the material of interest out of the centrifuge.
And in another aspect of the invention an annular centrifuge bowl includes a cup shape receptacle located above a central space surrounded by the bowl. The receptacle includes apertures which feed cleaning fluid into the central space to clean the surfaces of the bowl surrounding the central space.
Accordingly, it is an aspect of the present invention to provide an improved apparatus and methods for centrifuging materials in a vacuum environment.
Another object of the present invention is the provision of a centrifuge apparatus and methods for recovery of a protein of interest from the interstitial fluid of plant tissues, which apparatus includes a centrifuge bowl having a radially outwardly inclined outer wall for aiding the removal of the interstitial fluids from the centrifuge bowl.
And another object of the present invention is the provision of a centrifuge apparatus and methods having internal cleaning nozzles arranged for automated cleaning of the centrifuge apparatus between batches of material being processed.
Other and further objects, features and advantages of the present invention will be readily apparent to those skilled in the art upon a reading of the following disclosure when taken in conjunction with the accompanying drawings.